DESCRIPTION (Adapted from the abstract provided by the applicant): We propose to investigate the role that mutations in the mitochondrial adenine nucleotide translocator (ANT) genes play in neuromuscular degenerative diseases. The ANTs energetically link the mitochondria with the cytosol by exchanging mitochondrial ATP for cytosolic ADP. They are also thought to be an integral component of the mitochondrial permeability transition pore (mtPTP) and, therefore, central to apoptosis. Humans have three ANT isoform genes: the heart-muscle ANT1, the inducible ANT2, and the constitutive ANT3. Mouse has two ANT isoforms: Antl expressed in the skeletal muscle, heart, and brain and analogous to human ANT1, and Ant2 expressed in all tissue but skeletal muscle and analogous to human ANT2 plus ANT3. Mutations in human ANT1 have recently been shown to cause autosomal dominant progressive external opthalmoplegia (adPEO), a neuromuscular disease associated with multiple mitochondrial deletions. Previously, we generated an Antl knockout mouse which has similar features to adPEO and thus is an excellent model for ANT1 disease. Recently, we have also prepared a LoxP-Ant2-LoxP mouse permitting the tissue-specific elimination of Ant2. To further define the role of ANT defects in neuromuscular diseases, we propose four Specific Aims. Specific Aim 1 is to investigate the pathophysiology of ANT1 disease. We will determine the neuromuscular phenotype, physiological defects and Mitochip gene expression profiles of Ant1 +/- and -/- mice. Specific Aim 2 is to explore the nature of ANT2 and ANT3 disease. We will combine our LoxP-Ant2-LoxP mice with different Cre-promoter constructs to generate females that are Ant2 heterozygotes and animals that are Ant2-deficient in heart, cortex and cerebellum. These animals will be characterized for their phenotype, physiology and Mitochip gene expression profiles. We will also generate mice that are Ant1 and Ant2 null mutants in their livers and use the liver mitochondria to investigate the role of the ANTs in the mtPTP and in apoptosis. Specific Aim 3 takes advantage of the phenotypes exhibited by our Ant1 and Ant2 mouse mutants as guides to identify neuromuscular disease patients likely to harbor ANT defects. We will then use our isoform-specific antibodies and Mitochip expression profiles to screen for ANT defects and identify the mutant genes by ANT gene sequencing. Finally, in Specific Aim 4, we will use the Ant1 -/- mouse as a model system to develop an AAV gene therapy procedure for ANT1adPEO. These studies, upon completion, will provide critical new insights into the role of ANTs in mitochondrial function and neurodegenerative diseases.